PROJECT SUMMARY Despite advances in surgery and chemotherapy, ovarian cancer (OVCA) remains the most lethal gynecologic malignancy. The tumor microenvironment (TME) is a complex milieu of several types of cells, blood vessels and extracellular matrix proteins in which cancerous cells thrive. The cells that constitute most of the TME are fibroblasts, immune cells, endothelial cells and pericytes and are also collectively known as stroma. These cells become reactive and develop characteristics that support and even enhance tumor progression and metastasis due to proximity and constant interaction with cancer cells. Failure of traditional therapy is due to our limited understanding of how the TME can facilitate the rapid progression or recurrence of OVCA. Targeting reactive stromal cells is emerging as an attractive and viable therapy to regulate the channels of communication between stromal and cancer cells. To target non-autonomous mechanisms of cancer cell aberrations, the mechanistic underpinnings of reactive stroma vis a vis quiescent or normal stroma is required. Stromal cells such as cancer associated fibroblasts (CAFs), cancer associated mesothelial cells (CAMs), and cancer associated adipocytes (CAAs) in omental tissue have been shown to promote OVCA metastasis and growth. Although it has been recently shown that microenvironment can induce metabolic reprogramming in cancer cells, however, identification of stromal targets which make cancer cells vulnerable has remained challenging and elusive. We propose a previously unrecognized mechanism whereby metabolism of reactive stromal cells is reprogrammed through upregulated glutamine anabolic pathway. We first hypothesize that reactive stromal metabolism is altered from quiescent stroma, and is the driver for regulating cancer growth in its harsh microenvironment. Second, targeting this aberration could create metabolic vulnerability in cancer cells by disrupting the metabolic crosstalk between stromal and cancer cells. We will test these hypotheses in the proposed Aims. First, we will establish whether CAFs, CAAs, and CAMs promote OVCA cell proliferation by reprogramming glutamine (Gln) metabolism in cancer cells. We will validate upregulation of Gln anabolic pathway in reactive stromal cells compared to their normal counterparts through transcriptomic profiling. To elucidate metabolic reprogramming in reactive stromal cells we will use 13C-based metabolic flux analysis using stable isotope tracers to reveal metabolic vulnerabilities in stromal cells and unravel metabolic symbiosis between stromal and epithelial cells. Second, we will elucidate stroma-secreted Gln's role in maintaining OVCA cells' drug resistance. Our results will reveal an alternative modality in the treatment of recurrent OVCA. Third, we will determine the efficacy of targeting stromal Gln metabolism using orthotopic models of ovarian carcinoma and perform tracing of metabolic fates of different nutrients in tumors using in vivo tracer analysis in orthotopic models proposed for targeting stromal metabolism. In summary, our proposed study can lead to novel therapeutics targeting communication between cancer cells and their microenvironment.